GLP-1 derivatives are used in clinical trials to treat type II diabetes and obesity (Gallwitz B, Eur. Endocrinol., 2015; 11:21-5). GLP-1 induces multiple biological effects, for example, stimulation of insulin secretion, inhibition of glucagon secretion, inhibition of gastric emptying, inhibition of gastric and intestinal movement, as well as induction of loss of body weight (Lund A et al., Eur. J. Intern. Med., 2014; 25:407-14). One prominent characteristic of GLP-1 is its capability of stimulating the secretion of insulin without the risk of inducing hypoglycemia, which is one of the concern in insulin treatment and some other oral therapies that promote endogenous insulin secretion. Endogenous GLP-1 is degraded very quickly and its extremely short half-life limits the effectiveness of GLP-1 as a therapeutic peptide.
Currently, there are various ways to prolong the half-live of GLP-1 and its derivatives and maintain their biological activity at the same time (Verspohl E J, Pharmacol. Rev, 2012; 64:A-AX), including fusion of GLP-1 and its derivatives with an IgG Fc fragment or human serum albumin (HSA). The new method we have chosen is fusing GLP-1 with a full-length anti-GLP-1R antibody (IgG). The IgG can extend the in vivo half-life of its fusion partner and its own half-life in human is 21 days. In addition to maintaining the biological activity of GLP-1, a GLP-1R antibody fusion protein has advantageous stability provided by the IgG moiety. At the same time, the IgG moiety provides the GLP-1R antibody fusion protein molecular targeting properties, thus increasing the possibility of the interactions between GLP-1 and GLP-1R. Furthermore, the antibody molecule has lower immunogenicity than most of other commonly used fusion partners. As to a drug for a long-term or even life-long use, low immunogenicity is a necessity.
Fusion proteins are often produced by mammalian cell lines, for example, CHO, SP2/0 or NSO. It is noted in the present invention that, when CHO cells were used to produce a GLP-1R antibody fusion protein, the fusion protein was subjected to degradation by endogenous proteases or under certain cell culturing physicochemical conditions. The degradation process was faster and more apparent under low pH conditions, and on top of that, the GLP-1R antibody fusion protein was more prone to aggregation when pH was higher than 7. Furthermore, it was discovered in the present invention that under refrigeration conditions (2-8° C.) and in a single buffer solution (for example, a citrate buffer system), the solubility of the GLP-1R antibody fusion protein is too low to meet the requirements for therapeutic dosages. In the present invention, the problems mentioned above are solved through pH control, using a specific combination of certain excipients and a specific concentration of the GLP-1R antibody fusion protein.